In hypotrichous ciliates, all of the macronuclear DNA is in the form of low molecular weight molecules with an average size of -2200 base pairs. Total macronuclear DNA from four hypotrichs has been shown to have inverted terminal repeats by direct sequence analysis. In Oxytricha nova, Oxytricha sp., and Stylonychia pustulata, this terminal sequence may be written as 5'-C4A4C4A4C4 ... 3'-G4T4G4T4G4T4G4T4G4 ...In Euplotes aediculatus, the sequence is similar but differs in the lengths of the duplex region (28 base pairs) and of the putative 3' extension (14 base pairs). Also in Euplotes, a second common sequence of 5 base pairs (A:T:G-A-A) occurs internal to the terminal repeat and a 17-base-pair heterogeneous region:The length of the terminal repeat sequence for 0. nova was confirmed in cloned macronuclear DNA molecules.Ciliated protozoa of the order Hypotrichida have two types of nuclei-a small transcriptionally inactive (germinal) micronucleus and a large DNA-rich macronucleus, which contains the entire functional vegetative genome. Whereas the DNA of the micronucleus is large (>>150 X 10' daltons) and is contained in chromosomes, the transcriptionally active macronuclear genome is achromosomal; the DNA exists as a heterogeneous distribution of molecules ranging in size from 400 to ==20,000 base pairs (bp) with an average of 2200 bp (1, 2).The macronucleus is formed de novo from a diploid micronucleus after sexual conjugation. In brief, formation of the macronucleus involves the polytenization of micronuclear chromosomes, followed by fragmentation of polytene chromosomes at interbands and degradation of --95% of the DNA. The degradation phase eliminates all detectable repetitive DNA and most of the unique sequence DNA. What remains are small DNA molecules, which are extensively replicated to form the mature macronuclear genome (for review, see ref.3).This subset of the micronuclear DNA sequences that is retained in the macronucleus contains all of the genetic information (except for mitochondrial) for vegetative growth.The macronucleus contains --24,000 different DNA sequences, present at an average of 1000 copies per macronucleus. Each of these gene-sized molecules is a separate functional genetic unit. Denaturation of macronuclear DNA followed by a short period of reassociation leads to formation of singlestranded circles in more than 80% ofthe molecules as observed by electron microscopy. The single-stranded DNA is held in a circular configuration by a short duplex region. Therefore, all or nearly all of the gene-sized molecules of the macronucleus have an inverted terminal repeat (4). The sequence of this terminal repeat was shown to be the same in many ofthe molecules (5), although the sequence reported for the terminal repeat was not correct. We report here the correct sequence and show that the sequence forming the inverted terminal repeat is almost the same in four different hypotrich species. To establish the structure of the termini of native DNA molecules, we determined both the 3'-and 5'-en...

Macronuclear DNA s from three related hypotrichous ciliated protozoans were compared by agarose gel electrophoresis. Each was shown to be composed of DNA duplexes that yielded a unique pattern of bands overlying a continuous distribution of DNA sizes ranging from approximately 400 bp to approximately 20,000 bp. By EM, the number average molecular sizes for double-stranded DNA were 2,200 bp for Oxytricha sp., 2,514 bp for Stylonychia pustulata and 18,836 bp for Euplotes aediculatus. Contrary to previous reports we present evidence that the macronuclear DNA s in each of these three organisms lack single-stranded interruptions.

All of the genes in the macronucleus of Oxytricha sp. occur on physically separate "gene-sized" DNA molecules. We have inserted the DNA molecule that codes for rRNA into a bacterial plasmid in order to study its structure and function. Using restriction nuclease mapping and hybridization of 125I-rRNAs to gel separated DNA fragments we have determined that the intact rDNA is 8,140+/-50 base pairs (bp) in length. Reading from one end, the molecule consists of approximately 1,540 bp of non-coding DNA, approximately 2,100+/-50 bp that code for 19S rRNA, approximately 3,700+/-50 bp that code for 25 rTNA, and approximately 620+/-50 bp of non-coding DNDA. The 5.8S rRNA coding sequence (approximately 150 bp) occurs at one end of the 25S RNA coding region but which end is not known yet. All three rRNAs are encoded in the same strand of the DNA molecule, and transcription is in the order: 19S leads to 25S.

We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them.

Escherichia coli and Bacillus subtilis RNA polymerase have almost identical transcription specificities on bacteriophage SPO1 DNA when assayed in a coupled transcription-translation cell free system. SPO1-modified B. subtilis RNA polymerase has altered transcription specificity. It is shown that rifampicin-inhibited E. coli RNA polymerase can completely block transcription of SPO1 DNA by rifampicin resistant B. subtilis enzyme, whereas it has no effect on transcription by SPO1-modified B. subtilis RNA polymerase. We conclude that the new transcription by SPO1-modified RNA polymerase results from newly recognized promoters, rather than by elongation of transcripts which could also be made by B. subtilis vegetative RNA polymerase.